Sensitive Analysis of 33 Free Amino Acids in Serum, Milk, and Muscle by Ultra-High Performance Liquid Chromatography-Quadrupole-Orbitrap High Resolution Mass Spectrometry

Quantitative analysis of 33 free amino acids (AAs) in serum, milk, and muscle samples was achieved using ultra-high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The samples were extracted using methanol and derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate to enhance chromatographic retention. Thirty-three AAs were separated on a 1.7-μm BEH C18 column within 15 min, which is at least six times faster than that of an AA analyzer. Serum, milk, and muscle samples spiked with free AAs were tested in terms of linearity, sensitivity, repeatability, and recovery. Results indicated that recoveries for the AAs ranged from 77.7 to 124.8 % with relative standard deviations (RSD) less than 12.3 %. The lower limit of detection ranged from 0.003 to 0.68 μmol/L and 0.003 to 0.27 nmol/g for body fluid and muscle samples, respectively. Finally, the developed method was used for the determination of free AAs in actual serum, milk, and muscle samples with good results.